PROJECT SUMMARY/ABSTRACT Rotator cuff health relies on the functional attachment of cuff muscle and tendon to bone via the tendon- bone enthesis. The enthesis transmits muscle forces to the skeleton, and the structure and function of the enthesis is dependent on muscle loading. In the case of muscle or nerve injury at birth, the enthesis fails to properly form due to traction-induced muscle disuse of the shoulder at childbirth. Return of muscle function during muscle recovery in adolescents can predispose the enthesis to damage and rupture due to delayed enthesis formation during critical periods of postnatal maturation. The PI has recently shown the requirement for muscle loading during the growth and maturation of the enthesis, as well as for proper healing of the repaired rotator cuff. While it is understood that the muscle loading is required for the development of the enthesis, experimental approaches that induce controlled muscle activity during growth are non-existent. One potential way of controlling muscle activation is via the use of optogenetic tools, which control cell behavior via light-activation. Our laboratory is focused on identifying interventions, such as regulating muscle force, that promote the maturation and remodeling of the enthesis. This work will have implications for treating developmental disorders like muscle disuse, as we will develop approaches for understanding how muscle activation influences the maturation of the enthesis as well as how the adolescent enthesis adapts following muscle disuse and return to loading. In our first aim, we aim to develop an optogenetic mouse model for the localized activation of neonatal shoulder muscles. Using transgenic mice and cre-recombination, we will develop and validate a skeletal muscle-specific expression of channelrhodopsin-2 (hChR2)/YFP fusion protein for the activation of skeletal muscle following exposure to blue light (450-490nm). In our second aim, we will reactive rotator cuff muscles following disuse to regain enthesis morphology and mechanical function in an established model of neonatal muscle disuse. These findings will provide strong preliminary data for a future R01 proposal that will investigate the role of targeted muscle stimulation in a small animal model of muscle disuse. This research fits within the long-term goals of NICHD by aiming to ensure the health, well-being, and independence of adolescent individuals through optimized rehabilitation approaches in this difficult-to-treat population. By utilizing multidisciplinary approaches from developmental biology, biomechanics, engineering, and rehabilitation, the PI is poised to establish a strong and integrative research program in biomedical engineering.